Most human therapeutics have been discovered by screening natural products. Synthetic organic chemistry has made it possible to synthesize such natural products and derivatives thereof in large quantities, thus broadening the range of compounds that can be used clinically (Gates et al., J. Am. Chem. Soc. 74:1109-1110 (1952); Wipf et al., J. Am. Chem. Soc. 117:558-559 (1995); Nicolaou et al., Nature 392:264-269 (1998)). Synthetic methodology coupled with the outpouring of protein structural information has also allowed rational design of completely new therapeutic compounds (Gait et al., TIBTECH 13:430-438 (1995); Skulnick et al., J. Med. Chem. 40:1149-1164 (1997)). Similarly, the recent explosion in nucleic acid sequence information is providing a knowledge base for structure-based targeting of RNA. The first generation of such therapeutics consists of antisense nucleic acids that bind mRNA through Watson-Crick base-pairing and thereby regulate translation (Chrissey, Antisense Res. Dev. 1:65-113 (1991); Baserga et al., Ed. (1992) Antisense Strategies; Annals of the New York Academy of Sciences 660; New York Academy of Sciences: New York). Nucleic acids used for antisense therapeutics, typically between 15-20 nucleotides long, suffer from a number of disadvantages including high cost of synthesis (Wagner et al., Nature Biotechnology 14:840-844 (1996)), lack of specificity (Herschlag, Proc. Natl. Acad. Sci. U.S.A. 88:6921-6925 (1991)) and instability in vivo. Some of these disadvantages can be overcome by designing oligonucleotides in which the phosphodiester moiety is replaced by a more stable linking group. Earlier work by the present inventors has shown that short oligonucleotides in which the phosphodiesters are replaced by phosphoramidates bind as tightly, if not more tightly, to a complementary sequence.
Many opportunistic pathogens, in particular, fungal pathogens, have RNA elements that can serve as molecular targets for pharmacological intervention. Group I introns are one example of such an RNA element. Many pathogenic fungi have Group I introns in critical structural RNAs, for example, in ribosomal RNAs (rRNA). RNAs containing Group I introns undergo a process of self-splicing to remove the intron to produce a functional RNA. This self-splicing process of Group I introns is well known. For a review of the Group I intron splicing process, as well as a discussion of the properties of Group I introns in general, see Cech, “Self-Splicing of Group I Introns”, Ann. Rev. Biochem. 59:543 (1990). Group I introns contain a guanosine binding site and catalyze a reaction in which a guanosine (or a guanosine nucleotide) attacks the 5′ residue of the intron to produce 5′ exon and guanosine-intron-3′ exon intermediates, which then further react to yield linear guanosine-intron and the spliced 5′ exon-3′ exon product. During the self-splicing reaction, a region of the RNA at the 3′ end of the 5′ exon is thought to pair with a complementary sequence within the intron (the internal guide sequence or IGS) to align the 5′ splice site for reaction. FIG. 2 (right side panel A1 through C1) depicts this pairing and subsequent guanosine attack and cleavage followed by joining of the exons.
Pneumocystis carinii is an opportunistic pathogen that is a common cause of death in immunocompromised patients (Hughes, Annu. Rev. Med. 42:287-295 (1991); Steinberg, Science 266:1632-1634 (1994)). The large subunit ribosomal RNA (rRNA) precursor contains a Group I self-splicing intron (Testa et al., Biochemistry 36:15303-15314 (1997); Liu et al., Nucleic Acids Res. 20:3763-3772 (1992)) that provides a potential therapeutic target (Liu et al., (1992); Mei et al., Bioorg. Med. Chem. 5:1185-1195 (1997)) since self-splicing is required for assembly of active ribosomes (Nikolcheva et al., RNA 3:1016-1027 (1997)). Other pathogenic organisms, including Candida albicans (Mercure et al., Nucleic Acids Res. 21:6020-6027 (1993)) and Aspergillus nidulans (Netzker et al., Nucleic Acids Res. 10:4783-4790 (1982)), are also known to contain Group I introns, particularly within their rRNAs. Group I introns have not been found in humans to date. Earlier work of the present inventors showed that an oligonucleotide hexamer having a sequence that “mimics” the sequence of the putative 5′ exon guide sequence of a P. carinii ribosomal RNA Group I intron can tightly bind to a derived ribozyme through base-pairing and tertiary interaction. However, the ability of the mimic to compete for binding to the IGS with the endogenous 5′ exon guide sequence was not shown because the derived ribozyme used for this work did not contain the 5′ exon guide sequence that is endogenous to the P. carinii rRNA precursor.